As a method for optically analyzing blood cells in the blood such as red blood cell, white blood cell, platelet and the like, flow cytometry is known. Flow cytometry is a technique including irradiating a predetermined irradiation light as a beam light focused on blood cells in a sample solution (sample liquid) advancing through a flow channel, and analysis such as distinguishing, counting and the like of the blood cells from the resulting optical characteristics such as light scattering, light absorbance and the like (e.g., JP-A-H8(1996)-327529).
FIG. 8 is a sectional view of a configuration example of an apparatus for counting blood cells based on flow cytometry. In flow cytometry, as shown in FIG. 8, a sample solution M10 containing blood cells X10 flows through a flow channel 110, and an irradiation light L10 is irradiated from a light source device 200 through an optical system OP10 on a predetermined irradiation segment in the flow channel. Then, various optical characteristics such as the level of light absorption, level of scattering, level of fluorescence and the like, by each blood cell, with respect to a light (hereinafter to be also referred to as “transmitted light” for the sake of explanation) L20 produced when the irradiation light L10 hits blood cells X10, are measured by a light receiving device 300 through an optical system OP20. The size, kind, number, state and the like of the blood cells are identified from the measurement results. In FIG. 8, the optical systems OP10, OP20 are shown by blocks with a dot and dash line. In fact, a necessary number of optical parts such as lens (lenses) and the like are disposed on the optical path. The optical system OP10 on the light source device side contains a mask member (mentioned later) that forms the cross sectional shape (transverse sectional shape) of the irradiation light L10 into a predetermined shape. The mask member is also called a slit depending on the opening shape of the through-hole.
A part containing a flow channel configured to perform flow cytometry is also called a flow cell. The flow cell may be a single tube. In the apparatus of FIG. 8, the upstream side (lower side of the Figure) of a flow channel 110 has a double tube structure, wherein the flow of the sample solution M10 (containing blood cells X10) from an inner tube 120 is surrounded by a sheath flow from an outer tube 130 and enters into the flow channel 110. Due to this configuration, the flow of the sample solution becomes narrower and the blood cells X10 pass through the flow channel 110 one by one in an orderly manner, which permits irradiation of the irradiation light L10 on each blood cell in an irradiation segment. The wall containing the irradiation segment is transparent so that the irradiation light can penetrate the wall.
Furthermore, it may have a triple tube structure by adding an outer tube to the aforementioned double tube structure. In this case, the flow of a sample solution from the inner tube is surrounded by the first sheath flow, and the flow is further surrounded by the second sheath flow, as a result of which a flow with a suppressed turbulence enters into the irradiation segment.
Conventionally, the above-mentioned particle analysis apparatuses use a halogen lamp as a light source (part generating light) of a light source device (device including light source, electronic power supply, wiring circuit, and housing). However, the halogen lamp has a high heating (calorific) value, and has a problem of a deteriorated performance of measurement since it influences the optical system. Also, a light source device using the halogen lamp has a limitation on downsizing due to the size of the lamp itself. Moreover, since the halogen lamp has a comparatively short rating life, the lamp replacement requires time and cost.
When the analysis apparatus is simultaneously equipped with plural optical measurement systems such as fluorescence measurement and the like, the light from the light source device needs to be dispensed to each optical measurement system. Thus, the light quantity is insufficient for each optical measurement system. However, when the output of the halogen lamp is increased to compensate for the shortage of light quantity, the heating value also increases, and an adverse influence on the optical systems becomes more remarkable. In addition, since a cooling structure becomes necessary, downsizing of the optical systems becomes more difficult, and the cost of the apparatus as a whole also becomes problematically high.
To solve the above-mentioned problems of the halogen lamp, the present inventors studied use of a light emitting diode (hereinafter to be also referred to as an LED) as the light source of the light source device. However, when the LED was actually used as the light source of the apparatus for analyzing minute and fine particles (e.g., blood cells) based on flow cytometry, the following problem was newly found, which is specific to flow cytometry requiring irradiation of the light to an extremely small region.
The problem is that accurate measurement results cannot be obtained in flow cytometry, since the length of the irradiation segment in the flow channel is small (generally about 10 μm-1000 μm), when the LED light is focused on such small irradiation segment by the optical system, the electrode formed in the center of the light extraction surface of the LED forms an obstacle and lowers the strength of the irradiation light in the central part. The problem is more concretely explained in the following.
FIG. 9-FIGS. 11(a) and 11(b) are schematic showings facilitating understanding of the above-mentioned problem.
In FIG. 9, FIG. 10, the LED 210 is drawn large in size for the sake of explanation, thus showing the electrode 212 formed on the light extraction surface 211 of the LED. In the following, the electrode formed on the light extraction surface 211 is also referred to simply as “electrode”. The LED 210 is mounted on a substrate 220, with the light extraction surface 211 facing toward the flow channel. An electric conductor on the substrate 220, and an electric conductor wire for bonding to be connected to the electrode 212 are omitted. While a light L10 emitted from the LED 210 is drawn to be released solely from the light extraction surface 211 for the sake of explanation, it is in fact also released from a side surface of the LED, and sent in the outgoing direction by a reflection plate and the like (not shown).
On the optical path, lens OP110, mask member OP120, and lens OP130 are provided as the optical system OP10 on the light source device side, and a lens OP20 is provided as an optical system on the light receiving device 300 side. In the Figure, while each lens is shown as a block drawn with a dot and dash line for the sake of explanation, it is in fact also released from a side surface of the LED, and sent in the outgoing direction by a reflection plate and the like (not shown). In fact, many lenses such as combination lens wherein plural lenses are layered, and the like, are used as necessary.
The irradiation light L10 emitted from the LED 210 is formed by a through-hole OP121 of the mask member OP120 to have a rectangular cross sectional shape and irradiated onto the irradiation segment of the flow channel 110.
However, due to the presence of an electrode 212, as an obstacle, formed in the center of the light extraction surface of LED 210 as shown in FIG. 10, in the central part of the irradiation light L10 contains a part having low intensity of the light (hereinafter to be also referred to as low intensity part) L10a. In FIG. 10, the low intensity part L10a is hatched for the sake of explanation. The light in the low intensity part L10a has extremely low intensity as compared to that of the surrounding high intensity part (hereinafter to be also referred to as high intensity part), and the light intensity of the low intensity part is 0 unless the surrounding light sneaks in. The low intensity part is present in the center of the irradiation light, as a region having light intensity sharply decreased from that of the surrounding high intensity part.
FIGS. 11(a) and 11(b) show an irradiation light having a cross sectional shape formed by a mask member and irradiation of the shape-formed light on the irradiation segment of a flow channel. As shown in FIG. 11(a), an irradiation light L10 irradiated on a mask member OP120 is formed to have a cross sectional shape corresponding to the opening of the through-hole OP121 such as rectangle and the like. A light having a formed cross sectional shape is released via the optical system and, as shown in FIG. 11(b), irradiated on the irradiation segment e10 of the flow channel.
As clearly shown in FIG. 11(a), the main part of the center of the cross section of the irradiation light cut away by the through-hole OP121 is occupied by the low intensity part L10a, and the surrounding part is the high intensity part L10b. According to the study by the present inventors, the electrode 212 of LED 210 is focused as an image when the cross section of irradiation light L10 is observed near the entrance of the through-hole OP121. When the central part of such irradiation light L10 passes through the mask member, the main part of the center irradiation light L10 to be irradiated on the irradiation segment e10 becomes the low intensity part L10a, as shown in FIG. 11(b). As a result, a difference between the intensity of transmitted light before entry of particles X10 into the irradiation segment e10, and the intensity of transmitted light after entry of particles X10 into the irradiation segment e10 (particularly, the low intensity part L10a in the irradiation segment) becomes small. What particles have passed through the irradiation segment e10 is determined based on the changes in the received light intensity in a light receiving element. As mentioned above, however, changes in the received light intensity become small due to the presence of the low intensity part and accuracy and reliability of the determination are degraded. These are the above-mentioned problems found by the present inventors.
As for the above-mentioned problems caused by an electrode of LED, the present inventors considered utilization of high intensity part L10b on the outer side of the irradiation light L10 shown in FIG. 11(a). However, as shown in FIG. 10, a light emitting layer 213 of LED emits the strongest light from a part just beneath the electrode 212, and the emission intensity becomes weaker as it transversely gets farther from said part. Therefore, it was found that an irradiation light having sufficient intensity and uniform light intensity cannot be provided over the entire opening of the through-hole (i.e., whole irradiation segment) from the high intensity part outside the irradiation light L10. When a large difference exists in the intensity of the irradiation light in the irradiation segment, the accuracy and reliability of particle determination by the light receiving element are degraded, even though an irradiation light with sufficient intensity seems to be provided over the irradiation segment as a whole, similar to the case where the above-mentioned low intensity part is present. This is because the size of particles cannot be determined appropriately since the received light intensity that should be indicated is not shown when the particles enter a region having a low irradiation light intensity, as mentioned above.
The above-mentioned problems of a light source device in flow cytometry possibly occur similarly not only for counting apparatuses and blood cell classifying apparatuses targeting blood cells, but also apparatuses for analyzing various particles by flow cytometry.
The problem of the present invention is to provide a particle analysis apparatus capable of affording an irradiation light with sufficient intensity on the irradiation segment of the flow channel, permitting down-sizing, and further, having a light source capable of providing uniform light irradiation on the irradiation segment.